Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocyte

Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020)

Individual Recognition in Domestic Cattle (Bos taurus): Evidence from 2D-Images of Heads from Different Breeds

BACKGROUND: In order to maintain cohesion of groups, social animals need to process social information efficiently. Visual individual recognition, which is distinguished from mere visual discrimination, has been studied in only few mammalian species. In addition, most previous studies used either a small number of subjects or a few various views as test stimuli. Dairy cattle, as a domestic species allow the testing of a good sample size and provide a large variety of test stimuli due to the morphological diversity of breeds. Hence cattle are a suitable model for studying individual visual recognition. This study demonstrates that cattle display visual individual recognition and shows the effect of both familiarity and coat diversity in discrimination. [br/]METHODOLOGY/PRINCIPAL FINDINGS: We tested whether 8 Prim'Holstein heifers could recognize 2D-images of heads of one cow (face, profiles, (3/4) views) from those of other cows. Experiments were based on a simultaneous discrimination paradigm through instrumental conditioning using food rewards. In Experiment 1, all images represented familiar cows (belonging to the same social group) from the Prim'Holstein breed. In Experiments 2, 3 and 4, images were from unfamiliar (unknown) individuals either from the same breed or other breeds. All heifers displayed individual recognition of familiar and unfamiliar individuals from their own breed. Subjects reached criterion sooner when recognizing a familiar individual than when recognizing an unfamiliar one (Exp 1: 3.1+/-0.7 vs. Exp 2: 5.2+/-1.2 sessions; Z = 1.99, N = 8, P = 0.046). In addition almost all subjects recognized unknown individuals from different breeds, however with greater difficulty. [br/] CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that cattle have efficient individual recognition based on categorization capacities. Social familiarity improved their performance. The recognition of individuals with very different coat characteristics from the subjects was the most difficult task. These results call for studies exploring the mechanisms involved in face recognition allowing interspecies comparisons, including humans

Evaluation of a web-based intervention to reduce antibiotic prescribing for LRTI in six European countries: quantitative process analysis of the GRACE/INTRO randomised controlled trial.

To reduce the spread of antibiotic resistance, there is a pressing need for worldwide implementation of effective interventions to promote more prudent prescribing of antibiotics for acute LRTI. This study is a process analysis of the GRACE/INTRO trial of a multifactorial intervention that reduced antibiotic prescribing for acute LRTI in six European countries. The aim was to understand how the interventions were implemented and to examine effects of the interventions on general practitioners' (GPs') and patients' attitudes

CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II

The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2) and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II). AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response

Nitric Oxide Antagonizes the Acid Tolerance Response that Protects Salmonella against Innate Gastric Defenses

Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment.Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses

Endothelial progenitor cells display clonal restriction in multiple myeloma

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V(H)). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V(H )primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM

FAM5C Contributes to Aggressive Periodontitis

Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1β, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis

Dectin-1: a role in antifungal defense and consequences of genetic polymorphisms in humans

The clinical relevance of fungal infections has increased dramatically in recent decades as a consequence of the rise of immunocompromised populations, and efforts to understand the underlying mechanisms of protective immunity have attracted renewed interest. Here we review Dectin-1, a pattern recognition receptor involved in antifungal immunity, and discuss recent discoveries of polymorphisms in the gene encoding this receptor which result in human disease